Method of organ regeneration

ABSTRACT

It is intended to provide a method of regenerating an organ or a part thereof which comprises transplanting bone marrow or hematopoietic stem cells into an injured mammal or a mammal having an injured in organ or a part thereof; a method of treating injury: a method of producing an organ or a part thereof; and an organ or a part thereof produced by this method.

TECHNICAL FIELD

The present invention relates to a method of organ regeneration.

BACKGROUND ART

Regenerative medicine is a therapeutic method of curing organs ortissues lost in diseases or accidents utilizing artificially culturedcells or the like. This therapeutic method has less side effect thanexisting medicines, and yet is expected to be applicable to treatment ofincurable diseases such as Alzheimer's disease. It is said thatregeneration of skin is the most promising regeneration to be put intopractice. There has been reported a success in generating the structureof derm by separating fibroblast cells from derm, mass-culturing thosecells and seeding the resultant cells on a collagen sheet. If such asheet is transplanted in a patient with injury in the skin (such as burninjury), it is believed that the sheet will be integrated into thepatient's skin to thereby regenerate that skin.

Regeneration medicine using stem cells has also been tried. Stem cellsare undifferentiated cells with both self-proliferation capacity anddifferentiation capacity. They are sources from which tissues or organsdevelop, and are present in almost all organs or tissues. Among variousstem cells such as hematopoietic or neural stem cells, ES cells(embryonic stem cells) have high proliferative capacity and are capableof differentiating into almost all types of tissues. ES cells areprepared from early embryos (fertilized eggs) about 5 to 7 days afterfertilization in the case of human and about 3 to 4 days afterfertilization in the case of mouse. Since ES cells have capacity todifferentiate into various cells and high proliferation capacity, theirapplication to a new therapeutic method of regenerating lost cells(regenerative medicine) is expected. However, ES cells have two majorproblems. One is an ethical problem that they are obtained fromfertilized eggs. The other is that, at present stage, it is extremelydifficult to control ES cells so that they differentiate into onlynecessary cells. Under circumstances, the present inventors have chosenbone marrow-derived stem cells to further investigate into regenerativemedicine.

The liver is the only organ capable of wide-ranged regeneration. Untilrecently, it was believed that the regeneration of the liver is onlyperformed by hepatocytes or egg cells. It is known that oval cells(cholangiole cells) present in the Herring duct intercalated betweenbile capillaries in the liver tissue has bipotency to differentiate intohepatocytes and bile duct cells. Further, Petersen et al. revealed thatit is highly possible that bone marrow-derived cells generatehepatocytes after administration of carbon tetrachloride and allylalcohol. Lagasse et al. show that bone marrow transplantation into atyrosinemia model mouse causes differentiation of its hepatocytes,resulting in partial improvement of the liver function (see Lagasse E.et al., Nat. Med. 20006(11): 1229-1234).

However, it has not been proved that stem cells having thecharacteristic of oval cells were used in the regeneration of diseasedor damaged liver tissue. Besides, the following points have not yet beenelucidated: can oval cells really be stem cells in the liver tissue andplay a major role in the regeneration of the liver tissue; or to whatextent hematopoietic stem cells can re-constitute diseased liver tissueand what situation do they need to differentiate into hepatocytes orother cells constituting the liver?

DISCLOSURE OF THE INVENTION

The present invention aims at providing a method of regenerating organs,a method of treating organs and a method of preparing organs, as well asregenerated organs.

As a result of intensive and extensive researches toward the solution ofabove problems, the present inventors have found that bone marrowtransplantation into a mammal with an impairment makes it possible forthe organ with the impairment to regenerate at a high ratio. Thus, thepresent invention has been achieved.

The present invention relates to the following:

-   (1) A method of regenerating an organ or a part thereof, comprising    performing bone marrow transplantation or hematopoietic stem cell    transplantation in a mammal having an impairment in the organ or the    part thereof.-   (2) A method of treating an impairment, comprising performing bone    marrow transplantation or hematopoietic stem cell transplantation in    a mammal having the impairment in an organ or a part thereof and    thereby regenerating the organ or the part thereof.-   (3) A method of preparing an organ or a part thereof, comprising    performing bone marrow transplantation or hematopoietic stem cell    transplantation in a mammal having an impairment in the organ or the    part thereof, thereby regenerating the organ or the part thereof,    and recovering the resultant regenerated organ or the part thereof-   (4) An organ or a part thereof which has been regenerated by    performing bone marrow transplantation or hematopoietic stem cell    transplantation in a mammal having an impairment in the organ or the    part thereof.

In the methods described in (1) to (3) above and the organ or the partthereof described in (4) above, cells used in the bone marrowtransplantation may be bone marrow cells, and hematopoietic stem cellsused in the hematopoietic stem cell transplantation may be derived from,for example, peripheral blood or cord blood (e.g. peripheral blood stemcells or cord blood stem cells). With respect to the impairment,functional disorders or physical or chemical injuries may be enumerated,for example. The mammal is not particularly limited. For example, a newborn mammal may be used. The organ is at least one selected from thegroup consisting of liver, heart, brain, lung, kidney, intestine,pancreas, eye, bone and tooth.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1(A)-1(D) provide photographs showing regeneration of the liver.

FIGS. 2(A)-2(D) provide photographs showing regeneration of the liver atthe resected stump.

FIGS. 3(A)-3(D) provide photographs showing differentiation of a largenumber of bone marrow-derived cells and appearance of individualmyocardial cells in the endocardium.

FIG. 4 is a photograph showing the presence of about 4 to 6 myocardialcells in a piece of coronary cross section.

FIG. 5 is a photograph created by analyzing coronary cross sections forthe presence of individual myocardial cells and preparingthree-dimensional (3D) images. This photograph shows stereoscopicallythat a great number of bone marrow stem cell-derived myocardial cellsare distributed throughout the heart.

FIGS. 6(A)-6(I) provide photographs showing the results of staining ofmyocardial cells with connexin 43 and troponin 1c, and photographsshowing the results of observation of GFP positive myocytes by Nomarskiimaging.

FIG. 7 is a photograph showing regeneration of lung and bronchusepithelial cells in a bone marrow-derived manner.

FIGS. 8(A)-8(D) provide photographs showing regeneration of mesangialcells in the kidney.

FIGS. 9(A)-9(C) provide photographs showing regeneration of cells in thesmall intestine.

FIGS. 10(A)-10(B) provide photographs showing regeneration of osteocytesand osteoblast cells in the bone.

FIGS. 11(A)-11(B) provide photographs showing regeneration of thegingiva and the tooth.

FIGS. 12(A)-12(B) provide photographs showing regeneration of thesurface layer of the eye.

FIG. 13 is a photograph showing regeneration of the ectocornea of theeye.

FIG. 14 is a photograph showing regeneration of nerve cells in thebrain.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinbelow, the present invention will be described in detail.

The present invention demonstrates that, by performing bone marrowtransplantation or hematopoietic stern cell transplantation (e.g.peripheral blood hematopoietic stern cell transplantation) in a mammalhaving an impairment in an organ or a part thereof, hematopoietic stemcells are differentiated into those cells constituting the organ at ahigh ratio and in a wide range, resulting in the regeneration of theorgan.

Hitherto, it was believed that no division or regeneration occurs in theheart or the brain after birth. The present invention has succeeded forthe first time in preparing (regenerating) an organ by creating a mammalinjury model and performing hematopoietic stem cell transplantationtherein.

Further, once the organs of new born mice have matured, stem cells inindividual tissues (such as liver, heart, etc.) exhibit their functionsfully. Therefore, it is believed that in the regeneration of an injuredtissue, stem cells existing in that tissue play a major role in theregeneration rather than hematopoietic stem cells. The present inventorshave paid attention to the immature environment surrounding newborns. Inthe immediately after birth when organs in the body grow rapidly,proliferation of individual cells constituting tissues is remarkable. Inview of that hematopoietic stem cells transplanted in newborns will beutilized in tissues at a high probability and that newborns are verylikely to have immature environment with high plasticity, the presentinventors performed hematopoietic stem cell transplantation in newbornsand also created injury models to examine regeneration.

In order to put into practice the regenerative medicine using thepluripotency of hematopoietic stem cells, the present inventorsconsidered regeneration of the liver at first. For the purpose ofpreparing highly pure regenerated livers, the livers of newborns(recipients) were partially excised and then bone marrow transplantation(in particular, transplantation of stem cells in the bone marrow) wasperformed. Analysis of the livers of these recipients revealed that agreat number of hepatocytes have been differentiated from thedonor-derived hematopoietic stem cells. As a result, regeneration of theliver using hematopoietic stem cells has become possible. Thus, it hasbeen found that the differentiation capacity of hematopoietic stem cellscan be manifested even in patients with impairments. The presentinvention has been achieved based on such finding, and will lead todevelopment of regenerative medicine using hematopoietic stem cells invarious organs.

1. Mammals

Specific examples of mammals which may be used in the present inventioninclude pig, bovine, horse, monkey, dog, sheep, goat, rat and mouse.Among all, mouse is preferable because model animals are abundant andinbred lines are established. Pig is also preferable because it issuitable for application to practical regenerative medicine. Clinicalapplication to human is also possible. In this case, first, informedconsent must be obtained and then patients should be selected understrict control of doctors. Newborn mammals used in the present inventionare not particularly limited. Newborns preferably within 4 days afterbirth, more preferably within 2 days after birth, may be used.

2. Organs or Parts Thereof

In the present invention, the term “organ” refers to every tissue ororgan necessary for performing life activities in the body and includesparts (e.g. cells, tissues, etc.) of organs.

As organs, various tissues and organs in the digestive system,respiratory system, urinary system, genital organs, cardiovascularsystem, lymph system, sense organ system, central nerve system, skeletalsystem, and muscles may be enumerated, for example.

Specific examples of above organs include, but are not limited to, thefollowing.

Digestive system: oral cavity, throat, esophagus, stomach, smallintestine, large intestine, liver, pancreas

Respiratory system: trachea, bronchus, lung, pleura

Urinary system: kidney, ureter, urinary bladder

Genital organs: internal sex organs, external sex organs

Cardiovascular system: heart, artery, vein

Lymph system: lymph vessel, lymph node, spleen, thymus

Sense organ system: visual organs (eye: oculus, accessory ocular organs,etc.), hearing organs (eardrum)

Central nerve system: brain (cerebrum, diencephalon, mesencephalon,cerebellum), medulla, spinal cord

Skeletal system: skull, backbone, rib, sternum, bone of upper limb,humerus, bone of lower limb, femur, etc.

Muscles: skeletal muscle, smooth muscle, etc.

Others: skin, tooth, gingiva

3. Impairments in Organs

In the present invention, the term “impairment” refers to a functionaldisorder or a physical or chemical injury occurring in one of theabove-mentioned organs or a part thereof. The term “a part” used hereinmeans a certain amount of the relevant organ which can be injured orresected without making it impossible for the organ to maintain itsfunction. This amount varies depending on the organ. In the case of theliver, for example, 0.1-50%, preferably 10-30% of the total organ is the“part” that may be excised in the present invention. According to aninternational classification of impairments, “functional disorder” meansa problem in psychosomatic function or bodily structure, such as aremarkable variation or loss. This term encompasses a disease state inwhich abnormality has occurred in the above-mentioned organs or a partthereof. The term “physical or chemical injury” means a physical orchemical damage to an organ or a part thereof; such damage includesthose which are caused by excising with a surgical knife, pricking witha needle, picking and peeling off with tweezers, exposing to highconcentration oxygen, laser irradiation, and irradiation. Wounds whichoccur when tissue samples for biopsy have been taken are also includedin the “injury”.

Specific examples of injury include excision of hepatic lobes with asurgical knife in the liver; wall-penetrating injury caused by punctureinto the cardiac cavity in the heart; injury caused by direct prickingwith a needle around the ventricle in the brain; and injury to theepithelium in the lung caused by exposure to high concentration oxygen.Injuries may be given to these organs by conventional methods such assurgical operation, or operation using an endoscope or celoscope.

4. Bone Marrow Transplantation or Hematopoietic Stem CellTransplantation

Cell transplantations performed in the present invention are classifiedinto bone marrow transplantation, peripheral blood stem celltransplantation and cord blood stem cell transplantation, depending onthe cell transplanted. Therefore, cells which may be used in the presentinvention are bone marrow cells, peripheral blood stem cells and cordblood stem cells.

Bone marrow is a tissue present in the medullary cavity formed byosteoclast cells located inside of the bone tissue, and is a majorhematopoietic tissue. In bone marrow, progenitor cells for the entireblood cell lineage cells (erythrocytes, granulocytes,monocytes-macrophages, megakaryocytes-platelets, mast cells,lymphocytes) are present, and released into the peripheral blood whenthey have matured and differentiated. These progenitor cells are derivedfrom hematopoietic stem cell. Peripheral blood stem cells are releasedinto the blood after chemotherapy (administration of anti-cancer drugs),or after the administration of granulocyte-stimulating factor thatinduces increase in leukocytes. Cord blood stem cells are hematopoieticstem cells present in the blood in the umbilical cord.

When bone marrow transplantation is selected in the present invention,mammal-derived bone marrow cells useful in the transplantation includebone marrow cells from any mammal including human. In this case, thedonor (supplier of the bone marrow) may be either allogeneic orheterogeneic to the recipient (receiver of the bone marrow).

Bone marrow cells may be collected by conventional methods such as bonemarrow aspiration. The resultant bone marrow cells may be used as theyare, or suspension cells alone may be used. When suspension cells areused, bone marrow cells are suspended in a culture broth (animal cellculture medium preferably containing 10% fetal calf serum), seeded onplastic dishes and cultured. By these operations, adhesive cells areadhered to the dishes, making it possible to recover only suspensioncells. The thus obtained culture broth containing suspension cells iscentrifuged to recover only suspension cells. As an animal cell culturemedium, DMEM, RPMI-1640, HamF 12 broth or a mixture thereof may be used.In the present invention, it is also possible to transplant onlyhematopoietic stem cells or mesenchymal stem cells from bone marrow.These hematopoietic stem cells or mesenchymal stem cells are alsoincluded in the “bone marrow cells” in the bone marrow transplantationof the invention.

If transplantation of peripheral blood stem cells or cord blood stemcells is selected in the present invention, peripheral blood stem cellsand cord blood stem cells may be collected by known methods. Asmammal-derived stem cells to be used in the peripheral blood stem cellor cord blood stem cell transplantation, stem cells of any mammalincluding human may be used. Like in the case of bone marrowtransplantation, the donor may be either allogeneic or heterogeneic tothe recipient.

In the present invention, it is also possible to select CD34⁺ cells,CD34⁺ CD38⁻ cells, side population (SP) cells, monocytes or the likefrom bone marrow cells (including hematopoietic stem cells andmesenchymal stem cells in bone marrow), peripheral blood stem cells andcord blood stem cells, if desired or necessary.

For the separation of cells for use in the transplantation(hematopoietic stem cells) from bone marrow, peripheral blood or cordblood, cells may be labeled with known surface antigens and subjected tosorting by flowcytometry to thereby isolate necessary hematopoietic stemcells alone at a purity of 95% or more. For the separation ofmesenchymal stem cells from bone marrow cells, adhesive cells may beseparated in the above-described culture. Alternatively, separationmethods using antibodies are also possible. It is known that mesenchymalstem cells are close to ES cells in their capacity and that theydifferentiate into cells of bone, cartilage, fat, heart, nerve, liver,and so on. Thus, they are attracting attention as the “second omnipotentcell”. Peripheral blood stem cells may be obtained by apheresis.

The time period from a partial injury of an organ to transplantation maybe within the range from 0 to 1 week, preferably 0 to 96 hrs (4 days),more preferably 0 to 48 hrs (2 days).

The bone marrow cells or hematopoietic stem cells prepared as describedabove are transplanted in a mammal with an impairment or a recipient(mammal) in which a part of an organ has been injured in advance. Themethod of transplantation is as described below.

First, pretreatment is performed. Administration of a high dose ofanti-cancer drug or total body irradiation is carried out sometime inthe period from 48 hrs to up to immediately before the transplantation,to thereby almost completely destroy the bone marrow cells, etc. in therecipient. On the day of transplantation, the bone marrow fluid,peripheral blood stem cells, cord blood stem cells or the like suppliedby the donor is drip-fed into the vein of the recipient.

The recipient is kept under control in a clean room until normal bloodcomponents have been generated and its conditions have been stabilized.After the transplantation, the recipient may die early because ofrejection, GVH disease (graft-versus-host disease), severe infection,etc. Therefore, the progress is observed continuously and, if necessary,immunosuppressant, antibiotics, or the like is administered.

5. Regeneration of Organs

Organs are allowed to regenerate after the bone marrow transplantationor hematopoietic stem cell transplantation performed as described above.The term “regeneration” means that new cells, tissues or organs whichare composed of donor-derived cells are newly born from the injuredsite. Since regeneration periods vary depending on the organ, thepresence or absence of regeneration is appropriately observed and organsshowing regeneration are allowed to regenerate up to pre-determinedsizes. For example, if 30% of the liver has been resected the liver isallowed to regenerate for 48 hrs to several weeks. In this case, it isbelieved that transplanted stem cells are accumulated at the injuredsite because of the resection and continue differentiation andproliferation even after the liver has been regenerated. In the case ofthe heart, it is impossible to perform resection as performed in theliver. However, puncture into the cardiac cavity in newborns is believedto be a sufficient injury. The period necessary for the regeneration isfrom several hours to 2 weeks. In the regenerative process of the heart,it is preferable to examine whether or not stem cells have a definitemorphology of cardiac muscle or to examine the time period required forobtaining the morphology of cardiac muscle.

For testing whether or not an organ is derived from a donor, any of theknown techniques may be used. For example, donor's bone marrow cells maybe labeled with GFP (green fluorescence protein) or a similar materialand then whether the label is expressed in the relevant organ of therecipient may be observed. The judgment on whether or not individualorgans have been regenerated in a donor-derived manner may made byimmunostaining. This is a direct and sure method of discriminationthough the size of cells can be an indicator.

When the relevant organ has grown (regenerated) to a pre-determinedsize, the organ is allowed to take in the recipient if it is clinicalapplication. Alternatively, when an experimental animal was used, theorgan is removed from the animal by surgical operation or the like. Inthis case, it is not necessary to wait until the organ has regeneratedto a pre-determined size. A part of the tissue or cells may be recoveredin the course of regeneration, if necessary.

In the present invention, when the liver is regenerated, it is possibleto obtain from bone marrow-derived stem cells not only hepatocytes butalso hepatic stellate cells, Kupffer cells, endothelial cells, and bileduct cells. Besides, the regenerated part of the liver has donor-derivedcells and highly pure. The organ removed as described above is used inorgan transplantation, etc. in the donor (supplier of bone marrow in thecase of bone marrow transplantation).

Hereinbelow, the present invention will be described in more detail withreference to the following Examples. However, the technical scope of thepresent invention is not limited to these Examples.

EXAMPLE 1

Regeneration of Liver

(1) Mice

GFP (green fluorescent protein)-transgenic mice (hereinafter, called“GFP mice”) were prepared by ligating a DNA encoding GFP to Actinpromoter (Okabe M. et al., FEBS Lett. 1997; 407(3):313-319). These GFPmice were used as donors in bone marrow transplantation. GFP isexpressed in every type of cells in the body. Therefore, when cellsderived from these mice have been transplanted in recipients, it ispossible to identify donor-derived cells by detecting GFP positivecells.

C57/BL6 mice were purchased from Charles River Japan and used asrecipients in bone marrow transplantation.

Both mice were bred in animal facilities of Kyushu University underspecific control.

(2) Preparation of Bone Marrow Cells

Bone marrow cells to be transplanted were prepared from GFP mice.Briefly, after dissection of GFP mice, bone marrow cells were collectedfrom thighs and shinbones. The collected donor cells were passed througha 25-gauge needle and a 40 μm mesh filter repeatedly to thereby preparea single cell suspension. In order to isolate hematopoietic progenitorcells, cells were cultured at 4° C. for 30 min with antibodies such asB220, CD3, Gr-1, Mac-1 and TER119. Then, after washing with 2% fetalcalf serum (FCS)-containing PBS, bone marrow cells were cultured withsheep-anti-rat immuno-magnetic beads (sheep-anti-rat IgG conjugatedDynabeads; M-450 DYNAL Great Neck, N.Y.). Cells not binding to the beadswere collected for further separation of Sca-1(+) cells. Since Sca-1 isone of the most important markers for mouse hematopoietic stem cells,Lin(−)Sca-1(+) cells were separated from the total bone marrow cells ofGFP mice. Positive selection of Sca-1(+) cells was performed onrat-anti-mouse Sca-1 antibody-conjugated microbeads. The resultantLin(−)Sca-1(+) cells were suspended in 50 μl of PBS.

(3) Partial Liver Excision

Newborn mice (C57/BL6) within 24 hrs after birth were anesthetized byintraperitoneal injection of 200 μg of ketamine chloride. After making a1 cm incision in the skin, almost one half of the hepatic lobes (about10-25% of the whole liver) was removed. The skin and the peritoneum weresutured with nylon thread.

(4) Transplantation of Hematopoietic Stem Cells

Newborn mice (C57/BL6) were treated with 500 Gy total body irradiation.Within 6 hrs after the irradiation to the recipient newborn mice (within24 hrs after the liver excision), GFP mice-derived Lin(−) Sca-1(+) cellsprepared as described above (5000 cells) were transplanted into each ofthe newborn C57/BL6 mice through the facial vein.

(5) Experiment on Chimera Phenomenon of Hematopoietic Stem Cells

Two to eight months after the transplantation, peripheral blood wascollected from the retro-orbital venous plexus, and bone marrow cellswere collected from the lower limbs of recipient mice.

Donor mice-derived cells were detected as GFP positive cells using FACSCalibur (Becton Dickinson). For the lineage expression of donor-derivedcells, peripheral blood or bone marrow cells were stained with B220,CD3, Gr-1, Mac-1 and TER119.

(6) Analysis of Differentiation in Liver

Mice 3 to 60 days after the bone marrow transplantation wereanesthetized by isoflurane inhalation and euthanized by cervicaldislocation. Immediately after dissection, the liver was fixed in 4%paraformaldehyde (PFA) at room temperature for 30 min. The fixed tissuewas dehydrated with graded alcohol series and then sliced into sections50 μm thick using a vibratome. Also, the total tissue was fixed in 4%paraformaldehyde (PFA) at room temperature for 10 min and frozen in OCT(optical cutting temperature) compound (10.24% polyvinyl alcohol, 4.26%polyethylene glycol, 85.5% non-reactive ingredients). This sample wasused for preparing thin sections of 4-6 μm.

(7) Imunofluorescence

Tissue sections were treated as described below depending on thethickness. Sections 50 μm thick were stained with an antibody andincubated with a primary antibody overnight at 4° C. After washing withPBS twice in 2 hrs, the sections were reacted with a secondary antibodybound to Cy-3 (Jackson Immunoresearch).

Sections 4-6 μm thick were stained with an antibody and reacted with aprimary antibody for 1 hr at room temperature. After washing, the tissuesections were incubated with a secondary antibody bound to Cy-3.

Immunostaining was analyzed carefully under a confocal microscope(Olympus).

(8) Results

(i) Analysis of the Chimera Phenomenon

Lin(−) Sca-1(+) cells were transplanted in each of the newborn recipientmice, and the resultant chimera phenomenon in hematopoietic cells in therecipient mice was analyzed. Every recipient mouse exhibited a chimeraphenomenon of donor cell type in 70% or more of its hematopoietic cells.

Further, donor-derived cells were separated from recipient bone marrowcells and transplanted in secondary newborn recipient. This means thatbone marrow cells include hematopoietic stem cells havingself-regeneration capacity.

In order to identify the distribution and external appearance of GFPpositive cells, the liver was observed under a fluorescent microscope athigh magnifications and low magnifications immediately after dissection(FIG. 1). As a result, bone marrow stem cell-derived GFP positive cellswere distributed throughout the liver. This shows that the liverregenerated from the resected stump is derived from bone marrow stemcells at a high ratio. Although most of the GFP positive cells werespindle-shaped, several percent of donor cells appeared to behepatocytes morphologically. A large number of GFP positive,spindle-shaped cells were distributed around the central vein (FIG. 1).

In FIG. 1A, the object seen at the lower right corner of the field andemitting fluorescence is a regenerated hepatic lobe of the liver.Compared to other hepatic lobes, GFP is strongly positive in this lobe.FIG. 1B shows the external appearance of a regenerated liver. Theregenerated liver has external appearance similar to that of normalliver. FIGS. 1C and 1D show findings obtained by observing theregenerated liver highly enlarged.

(ii) Regeneration of Liver

The regenerated hepatic lobe exhibited intense GFP fluorescence at theresected stump (FIGS. 2A, 2C). In FIG. 2, panels A and C show thegathering of GFP positive cells at the resected stump of the liver, andpanels B and D show the staining of hepatocytes with albumin antibody.Panel C is a highly enlarged image of panel A, and panel D is a highlyenlarged image of panel B. As shown in FIG. 2, the regenerated lobe is adonor-derived normal hepatic lobe, and donor-derived cells were presentthere at a high purity. The results shown in panels B and D proved thatalbumin positive cells are present at a high ratio because cells presentyellow color and that they are functionally normal hepatocytes producingalbumin.

EXAMPLE 2

Regeneration of Heart

(1) Mice and Bone Marrow Cells

Breeding/administration of mice and preparation of bone marrow cellswere carried out in the same manner as in Example 1.

(2) Partial Injury in Heart

Recipient mice (C57/BL6) immediately to up to 3 days after birth werepricked with a 29-gauge needle to injure the cardiac muscle withoutopening the chest. The success of this manual technique could be easilyconformed by reverse flow of the blood in the cardiac cavity.

(3) Transplantation of Bone Marrow

Pretreatment (irradiation) of mice and transplantation of hematopoieticcells therein were carried out in the same manner as in Example 1.

(4) Analysis of Differentiation in Heart

(i) Preparation of Heart Tissue Samples

Recipient mice were anesthetized by isoflurane inhalation and theneuthanized by cervical dislocation. Immediately after dissection, theheart tissue was fixed in 4% paraformaldehyde (at room temperature for30 min). The fixed tissue was dehydrated with graded alcohol series andthen sliced into sections 50 μm thick using a vibratome (MicroslicerDTK-1000; DSK). Also, the total tissue was fixed in 4% paraformaldehyde(PFA) (at room temperature for 10 min) and frozen in OCT compound forpreparing thin sections of 4-6 μm. After sufficient freezing, the tissuewas sliced into sections 6 μm thick using Cryostat (model CM3050S;Leica).

(ii) Immunostaining

Heart sections were stained with the antibodies described below. Inorder to identify myocardial cells, heart sections were stained withtroponin 1C (connexin 43), sarcomeric actin, connexin 43 or Nk×2.5. Whenthick sections were stained with an antibody, individual sections wereincubated with a primary antibody at 4° C. overnight. Then, afterwashing twice with PBS in 2 hrs, the sections were reacted with asecondary antibody bound to Cy-3 (Jackson Immunoresearch).

When thin sections were stained with an antibody, individual sectionswere incubated with a primary antibody at room temperature for 1 hr.Then, after washing, the tissue sections were incubated with a secondaryantibody bound to Cy-3.

Immunostaining was analyzed under a confocal microscope (Olympus).

(5) Results

Lin(−) bone marrow cells (5×10⁵ cells) were transplanted in each of thenewborn recipient mice, and the chimerism in the recipient hematopoieticcells was analyzed 2 or 5 months after the transplantation. As a result,donor cell-type chimerism was observed in every recipient mouse in 70%or more of its hematopoietic cells. Further, donor-derived cells wereseparated from recipient bone marrow cells and transplanted in secondaryrecipient newborn mice. Donor-derived cells were also observed in thesecondary recipient mice. From these results, it can be said that thebone marrow cells took in the primary recipient mice includehematopoietic stem cells having self-regeneration capacity.

Further, 2 months after the cardiopuncture and the hematopoietic stemcell transplantation, chimerism and changes in differentiation in theheart tissue were analyzed.

First, the total tissue was observed under a fluorescence microscope atan excitation wave length of 488 nm. In the pericardium, GFP positivecells were identified along the coronary artery. When tissue sampleswere cut sagittally, GFP positive regions were found along myocardialfibers (FIG. 3). A large number of GFP positive cells were found in aspindle shape (FIG. 3). In FIG. 3, panel A is an image obtained when theendocardium was observed under a fluorescence stereoscopic microscope. Alarge number of GFP positive cells (bone marrow-derived cells) arerecognized. Panels B, C and D show those myocardial cells whichdefinitely have striations among GFP positive cells. When fluorescenceobservation was conducted again, similar results were obtained. Thus, itwas found that the results concerning GFP positive cells havereproducibility and that a large number of myocytes can be used as aheart injury model. Further, as shown in FIG. 4, a plurality ofdonor-derived myocardial cells were observed in one cross section (arrowmarks 1 to 4). When each of them was enlarged, they exhibited a definitemorphology of striated muscle (FIG. 4).

Further, as a result of immunofluorescence analysis of myocardial cells,a large number of donor-derived myocytes were observed from the apex ofthe heart to the entire heart corresponding to the injured plane (FIG.5). FIG. 5 is a stereoscopic image composed of 40 cross sections. Theleft ventricle is shown in the center. Scattered dots with yellow to redcolors represent bone marrow-derived myocardial cells (arrow marks inFIG. 5).

Immunological analysis was also performed by staining heart sectionswith connexin 43 and troponin 1c. As a result, individual antibodystaining patterns were observed (FIG. 6). In FIG. 6, upper panel C showsthe results of immunostaining with troponin 1c. Panel A shows theresults of GFP staining alone. Panel B is a composite of panels A and C.From these results, expression of myocardial cell specific markers wasclearly confirmed. Likewise, middle panel F in FIG. 6 shows the resultsof staining with connexin 43. Panel D shows GFP positive (i.e. derivedfrom transplanted bone marrow cells) myocardial cells. Panel E is acomposite of panels D and F. From these results, it was confirmed thatmyocardial cells are bone marrow-derived cells. This demonstrates that,after injury, some types of myocardial cells may be differentiated frombone marrow-derived stem cells.

In order to further perform morphological analysis, GFP positivemyocardial cells were observed by Nomarski imaging (lower panels in FIG.6). The lower panels in FIG. 6 show relationships between the contoursof GFP positive cells and surrounding cells. From these panels, thecontours have become clear, and it has been found that donor-derivedbone marrow cells are incorporated surely and normally as myocardialcells.

From what have been described so far, it was confirmed that thosemyocardial cells are surely bone marrow derived cells morphologicallyand immunologically.

EXAMPLE 3

Regeneration of Other Organs

(1) Mice and Bone Marrow Cells

Breeding/administration of mice and preparation of bone marrow cellswere carried out in the same manner as in Example 1. Pretreatment(irradiation) of mice and transplantation of hematopoietic cells (bonemarrow transplantation) therein were also carried out in the same manneras in Example 1.

(2) Creation of Injury Models

(2-1) Injury Model of the Lung

Alveolar epithelial cells were injured by exposure to high concentrationoxygen or by administration of LPS (endotoxin).

(2-2) Injury Model of Kidney

Mesangial cells were injured with anti-Thy-1 antibody.

(2-3) Injury Model of Small Intestine

Radiation enteritis was induced by irradiation.

(2-4) Injury Model of Bone

Irradiation was performed.

(2-5) Injury Model of Tooth and Gingiva

The gingiva was directly injured with a needle or cells were poured intothe gingival.

(2-6) Injury Model of Eye

Irradiation was performed.

(2-7) Injury Model of Brain

Recipient mice (C57/BL6) immediately to up to 3 days after birth wereanesthetized, and then donor-derived cells were transplanted in themaround the ventricles.

(3) Analysis of Differentiation

Differentiation and regeneration of the following cells were examined:bronchial epithelial cells for the lung; mesangial cells for the kidney,epithelioid cells for the small intestine; cortical bone for the bone;dental surface and gingival cells for the tooth and the gingiva; eyesurface and parenchyma of cornea for the eye; and cells showing themorphologies of neuron and glia for the brain.

(4) Results

(4-1) Lung

It was shown that both pulmonary and bronchial epithelial cells areregenerated in a donor's bone marrow-derived manner. Double stainingwith cytokeratin confirmed that GFP positive cells are epithelial cells(FIG. 7).

(4-2) Kidney

In FIG. 8, panel A is a photograph where three GFP positive cells wereobserved. Panel B shows the results of staining with collagen 4. Panel Cis a composite of panels A and B; the three cells were identified asmesangial cells in the kidney. Panel D shows identification ofdonor-derived cells in the same manner as described above at a differentsite.

(4-3) Small Intestine

Donor-derived epithelioid cells were regenerated near the cavity of theintestinal tract (FIG. 9). In FIG. 9, panel A is a photograph where GFPpositive cells were observed. Panel B shows the results of staining withpan-cytokeratin. Panel C is a composite of panels A and B.

(4-4) Bone

Donor-derived cells were observed in the cortical bone (FIG. 10). FIG.10 provides photographs showing the cortical bone and the medullarycavity. Panel A is slightly enlarged and panel B is highly enlarged.“OC” represents osteocytes and “OB” osteoblast cells.

(4-5) Tooth and Gingiva

Regeneration was also recognized in the tooth and the gingiva (FIG. 11).In FIG. 11, panel A shows gingival cells and panel B shows cells nearthe dental surface. The mark (G) indicates GFP positive cells in thegingiva.

(4-6) Eye

Regeneration was recognized on the surface of the eye and in cornealepithelial cells (FIGS. 12 and 13). In FIG. 12, panel A shows a controlwhere bone marrow transplantation was not performed; GFP fluorescence isnot observed here. Panel B shows the eye of a recipient who receivedbone marrow transplantation; GFP fluorescence was recognized. FIG. 13shows cells within the cornea; donor-derived cells (GFP fluorescence)were recognized.

(4-7) Brain

Regeneration of cells having a neurite-like construct was recognized(FIG. 14).

As a result of analysis in the brain, it was found that nerve cellsderived from donor's bone marrow are differentiated.

INDUSTRIAL APPLICABILITY

A method for regenerating tissues is provided by the present invention.According to the method of the present invention, an organ can beregenerated by performing hematopoietic cell transplantation in a mammalhaving impairment in the organ. Besides, by giving an injury to anorgan, it is possible to construct at the injured sitetissue-constituting cells derived from bone marrow or the like. Further,the thus constructed cells may be collected and used in a therapeuticmethod where an artificial tissue is transplanted in a patient. Stillfurther, by creating disease animal models by injuring animals, it isalso possible to replace the injured site and peripheral regions thereofwith normal cells derived from bone marrow or the like. Therefore, themethod of the present invention is useful in still wide-rangedregenerative medicine.

1. A method of regenerating an organ or a part thereof, comprisingperforming bone marrow transplantation or hematopoietic stem celltransplantation in a mammal having an impairment in the organ or thepart thereof.
 2. The method according to claim 1, wherein thehematopoietic stem cell is peripheral blood hematopoietic stem cell orcord blood hematopoietic stem cell.
 3. A method of treating animpairment, comprising performing bone marrow transplantation orhematopoietic stem cell transplantation in a mammal having theimpairment in an organ or a part thereof and thereby regenerating theorgan or the part thereof.
 4. The method according to claim 3, whereinthe hematopoietic stem cell is peripheral blood hematopoietic stem cellor cord blood hematopoietic stem cell.
 5. A method of preparing an organor a part thereof, comprising performing bone marrow transplantation orhematopoietic stem cell transplantation in a mammal having an impairmentin the organ or the part thereof, thereby regenerating the organ or thepart thereof, and recovering the resultant regenerated organ or the partthereof.
 6. The method according to claim 5, wherein the hematopoieticstem cell is peripheral blood hematopoietic stem cell or cord bloodhematopoietic stem cell.
 7. The method according to claim 1, wherein theimpairment is a functional disorder of a physical or chemical injury. 8.The method according to claim 1, wherein the regenerated organ or thepart thereof is derived from a donor.
 9. The method according to claim1, wherein the mammal is a new born mammal.
 10. The method according toclaim 1, wherein the organ is at least one selected from the groupconsisting of liver, heart, brain, lung, kidney, intestine, pancreas,eye, bone and tooth.
 11. An organ or a part thereof which has beenregenerated by performing bone marrow transplantation or hematopoieticstem cell transplantation in a mammal having an impairment in the organor the part thereof.
 12. The organ or the part thereof according toclaim 11, wherein the hematopoietic stem cell is peripheral bloodhematopoietic stem cell or cord blood hematopoietic stem cell.
 13. Theorgan or the part thereof according to claim 11, wherein the impairmentis a functional disorder of a physical or chemical injury.
 14. The organor the part thereof according to claim 11, wherein the regenerated organor the part thereof is derived from a donor.
 15. The organ or the partthereof according to claim 11, wherein the mammal is a new born mammal.16. The organ or the part thereof according to claim 11, wherein theorgan is at least one selected from the group consisting of liver,heart, brain, lung, kidney, intestine, pancreas, eye, bone and tooth.17. The method according to claim 3, wherein the impairment is afunctional disorder of a physical or chemical injury.
 18. The methodaccording to claim 5, wherein the impairment is a functional disorder ofa physical or chemical injury.
 19. The method according to claim 3,wherein the regenerated organ or the part thereof is derived from adonor.
 20. The method according to claim 5, wherein the regeneratedorgan or the part thereof is derived from a donor.
 21. The methodaccording to claim 3, wherein the mammal is a new born mammal.
 22. Themethod according to claim 5, wherein the mammal is a new born mammal.23. The method according to claim 3, wherein the organ is at least oneselected from the group consisting of liver, heart, brain, lung, kidney,intestine, pancreas, eye, bone and tooth.
 24. The method according toclaim 5, wherein the organ is at least one selected from the groupconsisting of liver, heart, brain, lung, kidney, intestine, pancreas,eye, bone and tooth.